Effects of dual inhibition of AKT and ERK1/2 pathways on endometrial pro-inflammatory, hormonal, and epigenetic microenvironment in endometriosis.

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. The prevalence of endometriosis is ~5-10% in reproductive-age women, increasing to 20-30% in women with subfertility. The current anti-estrogen therapies can be prescribed only for a short time because of the undesirable side effects on menstruation, pregnancy, bone health, and failure to prevent a recurrence. The causes of endometriosis-associated infertility are multifactorial and poorly understood. The objective of the present study was to determine the inhibitory effects of AKT and/or ERK1/2 pathways on the microenvironment of the endometrium in a xenograft mouse model of endometriosis of human origin. Results indicate that dual inhibition of AKT and ERK1/2 pathways, but not inhibition of either AKT or ERK1/2 pathway, suppresses the growth of the endometriotic lesions in vivo. Dual inhibition of AKT and ERK1/2 pathways suppresses the production of proinflammatory cytokines, decreases E2 biosynthesis and signaling, and restores progesterone receptor-B signaling components in the epithelial and stromal cells of the endometrium in a cell-specific manner. These results together suggest that dual inhibition of AKT and ERK1/2 pathways suppresses the estrogen-dominant state and concomitantly increases the progesterone-responsive state of the endometrium. Therefore, dual inhibition of AKT and ERK1/2 pathways could emerge as long-term nonsteroidal therapy for endometriosis.

Intra- and Inter-Species Variability in Urinary N7-(1-Hydroxy-3-buten-2-yl)guanine Adducts Following Inhalation Exposure to 1,3-Butadiene.

1,3-Butadiene is a known carcinogen primarily targeting lymphoid tissues, lung, and liver. Cytochrome P450 activates butadiene to epoxides which form covalent DNA adducts that are thought to be a key mechanistic event in cancer. Previous studies suggested that inter-species, -tissue, and -individual susceptibility to adverse health effects of butadiene exposure may be due to differences in metabolism and other mechanisms. In this study, we aimed to examine the extent of inter-individual and inter-species variability in the urinary N7-(1-hydroxy-3-buten-2-yl)guanine (EB-GII) DNA adduct, a well-known biomarker of exposure to butadiene. For a population variability study in mice, we used the collaborative cross model. Female and male mice from five strains were exposed to filtered air or butadiene (590 ppm, 6 h/day, 5 days/week for 2 weeks) by inhalation. Urine samples were collected, and the metabolic activation of butadiene by DNA-reactive species was quantified as urinary EB-GII adducts. We quantified the degree of EB-GII variation across mouse strains and sexes; then, we compared this variation with the data from rats (exposed to 62.5 or 200 ppm butadiene) and humans (0.004-2.2 ppm butadiene). We show that sex and strain are significant contributors to the variability in urinary EB-GII levels in mice. In addition, we find that the degree of variability in urinary EB-GII in collaborative cross mice, when expressed as an uncertainty factor for the inter-individual variability (UFH), is relatively modest (≤threefold) possibly due to metabolic saturation. By contrast, the variability in urinary EB-GII (adjusted for exposure) observed in humans, while larger than the default value of 10-fold, is largely consistent with UFH estimates for other chemicals based on human data for non-cancer endpoints. Overall, these data demonstrate that urinary EB-GII levels, particularly from human studies, may be useful for quantitative characterization of human variability in cancer risks to butadiene.

Lung metabolome of 1,3-butadiene exposed Collaborative Cross mice reflects metabolic phenotype of human lung cancer.

1,3-Butadiene (BD) exposure is known to cause numerous adverse health effects, including cancer, in animals and humans. BD is metabolized to reactive epoxide intermediates, which are genotoxic, but it is not well know what other effects BD has on cellular metabolism. We examined the effects of exposure to BD on the mouse lung metabolome in the genetically heterogeneous collaborative cross outbred mouse model. Mice were exposed to 3 concentra-tions of BD for 10 days (2, 20, and 200 ppm), and lung tissues were analyzed using high-resolution mass spectrometry-based metabolomics. As compared to controls (0 ppm BD), BD had extensive effects on lung metabolism at all concentrations of exposure, including the lowest concentration of 2 ppm, as reflected by reprogramming of multiple metabolic pathways. Metabolites participating in glycolysis and the tricarboxylic acid cycle were elevated, with 8 out of 10 metabolites demonstrating a 2 to 8-fold increase, including the oncometabolite fumarate. Fatty acid levels, sphingosine, and sphinganine were decreased (2 to 8-fold), and fatty acyl-CoAs were significantly increased (16 to 31-fold), suggesting adjustments in lipid metabolism. Furthermore, metabolites involved in basic amino acid metabolism, steroid hormone metabolism, and nucleic acid metabolism were significantly altered. Overall, these changes mirror the metabolic alterations found in lung cancer cells, suggesting that very low doses of BD induce metabolic adaptations that may prevent or promote adverse health effects such as tumor formation.

Reactivation of BMP signaling by suboptimal concentrations of MEK inhibitor and FK506 reduces organ-specific breast cancer metastasis.

TGFß-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased TGFß-SMAD3 and a reduced BMP-SMAD1/5 targeted gene expression signature correlated with shortened distant metastasis free survival and overall survival of patients. At molecular levels, we discovered that TGFß abolished BMP-induced SMAD1/5 activation in the highly-invasive breast cancer MDA-MB-231 cells, but to a less extent in the non-invasive cancer and normal breast cells. This suggests an inverse correlation between BMP signaling and invasiveness of tumor cells and TGFß signaling acts in a double whammy fashion in driving cancer invasion and metastasis. Sustained ERK activation by TGFß was specifically observed in MDA-MB-231 cells, and MEK inhibitor (MEKi) treatment restored BMP-SMAD1/5 signaling while not affecting SMAD2/3 activation. FK506 potently activated BMP, but not TGFß signaling in breast cancer cells. MEKi or FK506 alone inhibited MDA-MB-231 extravasation in a zebrafish xenograft cancer model. Importantly, when administrated at suboptimal concentrations MEKi and FK506 strongly synergized in promoting BMP-SMAD1/5 signaling and inhibiting cancer cell extravasation. Furthermore, this combination of suboptimal concentrations treatment in a mouse tumor model resulted in real-time reduction of BMP-SMAD1/5 signaling in live tumors, and consequently potently inhibited tumor self-seeding, liver and bone metastasis, but not lung and brain metastasis. Mechanistically, it is the first time to identify BMP-SMAD1/5 signaling as an underlying molecular driver for organ-specific metastasis. Combining of MEKi and FK506, or their analogues, may be explored for clinical development of breast cancer.

Synergistic effects of a cremophor EL drug delivery system and its U0126 cargo in an ex vivo model.

Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ETB) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K+ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system.

Stimulating brain recovery after stroke using theranostic albumin nanocarriers loaded with nerve growth factor in combination therapy.

For many years, delivering drug molecules across the blood brain barrier has been a major challenge. The neuropeptide nerve growth factor is involved in the regulation of growth and differentiation of cholinergic neurons and holds great potential in the treatment of stroke. However, as with many other compounds, the biomolecule is not able to enter the central nervous system. In the present study, nerve growth factor and ultra-small particles of iron oxide were co-encapsulated into a chemically crosslinked albumin nanocarrier matrix which was modified on the surface with apolipoprotein E. These biodegradable nanoparticles with a size of 212 ±â€¯1 nm exhibited monodisperse size distribution and low toxicity. They delivered NGF through an artificial blood brain barrier and were able to induce neurite outgrowth in PC12 cells in vitro. In an animal model of stroke, the infarct size was significantly reduced compared to the vehicle control. The combination therapy of NGF and the small-molecular MEK inhibitor U0126 showed a slight but not significant difference compared to U0126 alone. However, further in vivo evidence suggests that successful delivery of the neuropeptide is possible as well as the synergism between those two treatments.

Dual inhibition of ERK1/2 and AKT pathways is required to suppress the growth and survival of endometriotic cells and lesions.

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. Current hormonal therapies targeting estrogen can be prescribed only for a short time. It indicates a need for non-hormonal therapy. ERK1/2 and AKT pathways control several intracellular signaling molecules that control growth and survival of cells. Objectives of the present study are to determine the dual inhibitory effects of ERK1/2 and AKT pathways: (i) on proliferation, survival, and apoptosis of human endometrioitc epithelial cells and stromal cells in vitro; (ii) on growth and survival of endometrioitc lesions in vivo in xenograft mouse model of endometriosis of human origin; and (iii) establish the associated ERK1/2 and AKT downstream intracellular signaling modules in the pathogenesis of endometriosis. Our results indicated that combined inhibition of ERK1/2 and AKT pathways highly decreased the growth and survival of human endometriotic epithelial cells and stromal cells in vitro and suppressed the growth of endometriotic lesions in vivo compared to inhibition of either ERK1/2 or AKT pathway individually. This cause-effect is associated with dysregulated intracellular signaling modules associated with cell cycle, cell survival, and cell apoptosis pathways. Collectively, our results indicate that dual inhibition of ERK1/2 and AKT pathways could emerge as potential non-hormonal therapy for the treatment of endometriosis.

Decreased microRNA-182-5p helps alendronate promote osteoblast proliferation and differentiation in osteoporosis via the Rap1/MAPK pathway.

Osteoporosis (OP) is a serious health problem that contributes to osteoporotic structural damage and bone fragility. MicroRNAs (miRNAs) can exert important functions over bone endocrinology. Therefore, it is of substantial significance to clarify the expression and function of miRNAs in bone endocrine physiology and pathology to improve the potential therapeutic value for metabolism-related bone diseases. We explored the effect of microRNA-182-5p (miR-182-5p) on osteoblast proliferation and differentiation in OP rats after alendronate (ALN) treatment by targeting adenylyl cyclase isoform 6 (ADCY6) through the Rap1/mitogen-activated protein kinase (MAPK) signaling pathway. Rat models of OP were established to observe the effect of ALN on OP, and the expression of miR-182-5p, ADCY6 and the Rap1/MAPK signaling pathway-related genes was determined. To determine the roles of miR-182-5p and ADCY6 in OP after ALN treatment, the relationship between miR-182 and ADCY6 was initially verified. Osteoblasts were subsequently extracted and transfected with a miR-182-5p inhibitor, miR-182-5p mimic, si-ADCY6 and the MAPK signaling pathway inhibitor U0126. Cell proliferation, apoptosis and differentiation were also determined. ALN treatment was able to ease the symptoms of OP. miR-182-5p negatively targeted ADCY6 to inhibit the Rap1/MAPK signaling pathway. Cells transfected with miR-182 inhibitor decreased the expression of ALP, BGP and COL I, which indicated that the down-regulation of miR-182-5p promoted cell differentiation and cell proliferation and inhibited cell apoptosis. In conclusion, the present study shows that down-regulated miR-182-5p promotes the proliferation and differentiation of osteoblasts in OP rats through Rap1/MAPK signaling pathway activation by up-regulating ADCY6, which may represent a novel target for OP treatment.

Effects of Sunitinib and Other Kinase Inhibitors on Cells Harboring a PDGFRB Mutation Associated with Infantile Myofibromatosis.

Infantile myofibromatosis represents one of the most common proliferative fibrous tumors of infancy and childhood. More effective treatment is needed for drug-resistant patients, and targeted therapy using specific protein kinase inhibitors could be a promising strategy. To date, several studies have confirmed a connection between the p.R561C mutation in gene encoding platelet-derived growth factor receptor beta (PDGFR-beta) and the development of infantile myofibromatosis. This study aimed to analyze the phosphorylation of important kinases in the NSTS-47 cell line derived from a tumor of a boy with infantile myofibromatosis who harbored the p.R561C mutation in PDGFR-beta. The second aim of this study was to investigate the effects of selected protein kinase inhibitors on cell signaling and the proliferative activity of NSTS-47 cells. We confirmed that this tumor cell line showed very high phosphorylation levels of PDGFR-beta, extracellular signal-regulated kinases (ERK) 1/2 and several other protein kinases. We also observed that PDGFR-beta phosphorylation in tumor cells is reduced by the receptor tyrosine kinase inhibitor sunitinib. In contrast, MAPK/ERK kinases (MEK) 1/2 and ERK1/2 kinases remained constitutively phosphorylated after treatment with sunitinib and other relevant protein kinase inhibitors. Our study showed that sunitinib is a very promising agent that affects the proliferation of tumor cells with a p.R561C mutation in PDGFR-beta.

STK33 alleviates gentamicin-induced ototoxicity in cochlear hair cells and House Ear Institute-Organ of Corti 1 cells.

Serine/threonine kinase 33 (STK33), a member of the calcium/calmodulin-dependent kinase (CAMK), plays vital roles in a wide spectrum of cell processes. The present study was designed to investigate whether STK33 expressed in the mammalian cochlea and, if so, what effect STK33 exerted on aminoglycoside-induced ototoxicity in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. Immunofluorescence staining and western blotting were performed to investigate STK33 expression in cochlear hair cells (HCs) and HEI-OC1 cells with or without gentamicin treatment. CCK8, flow cytometry, immunofluorescence staining and western blotting were employed to detect the effects of STK33 knockdown, and/or U0126, and/or N-acetyl-L-cysteine (NAC) on the sensitivity to gentamicin-induced ototoxicity in HEI-OC1 cells. We found that STK33 was expressed in both mice cochlear HCs and HEI-OC1 cells, and the expression of STK33 was significantly decreased in cochlear HCs and HEI-OC1 cells after gentamicin exposure. STK33 knockdown resulted in an increase in the cleaved caspase-3 and Bax expressions as well as cell apoptosis after gentamicin damage in HEI-OC1 cells. Mechanistic studies revealed that knockdown of STK33 led to activated mitochondrial apoptosis pathway as well as augmented reactive oxygen species (ROS) accumulation after gentamicin damage. Moreover, STK33 was involved in extracellular signal-regulated kinase 1/2 pathway in primary culture of HCs and HEI-OC1 cells in response to gentamicin insult. The findings from this work indicate that STK33 decreases the sensitivity to the apoptosis dependent on mitochondrial apoptotic pathway by regulating ROS generation after gentamicin treatment, which provides a new potential target for protection from the aminoglycoside-induced ototoxicity.

Noonan syndrome-causing SHP2 mutants impair ERK-dependent chondrocyte differentiation during endochondral bone growth.

Growth retardation is a constant feature of Noonan syndrome (NS) but its physiopathology remains poorly understood. We previously reported that hyperactive NS-causing SHP2 mutants impair the systemic production of insulin-like growth factor 1 (IGF1) through hyperactivation of the RAS/extracellular signal-regulated kinases (ERK) signalling pathway. Besides endocrine defects, a direct effect of these mutants on growth plate has not been explored, although recent studies have revealed an important physiological role for SHP2 in endochondral bone growth. We demonstrated that growth plate length was reduced in NS mice, mostly due to a shortening of the hypertrophic zone and to a lesser extent of the proliferating zone. These histological features were correlated with decreased expression of early chondrocyte differentiation markers, and with reduced alkaline phosphatase staining and activity, in NS murine primary chondrocytes. Although IGF1 treatment improved growth of NS mice, it did not fully reverse growth plate abnormalities, notably the decreased hypertrophic zone. In contrast, we documented a role of RAS/ERK hyperactivation at the growth plate level since 1) NS-causing SHP2 mutants enhance RAS/ERK activation in chondrocytes in vivo (NS mice) and in vitro (ATDC5 cells) and 2) inhibition of RAS/ERK hyperactivation by U0126 treatment alleviated growth plate abnormalities and enhanced chondrocyte differentiation. Similar effects were obtained by chronic treatment of NS mice with statins. In conclusion, we demonstrated that hyperactive NS-causing SHP2 mutants impair chondrocyte differentiation during endochondral bone growth through a local hyperactivation of the RAS/ERK signalling pathway, and that statin treatment may be a possible therapeutic approach in NS.

Fabrication of poly (butadiene-block-ethylene oxide) based amphiphilic polymersomes: An approach for improved oral pharmacokinetics of Sorafenib.

Sorafenib (SFN), a hydrophobic anticancer drug, has several limitations predominantly poor aqueous solubility and hepatic first-pass effect, limiting its oral delivery that results into several other complications. Present study aims to develop Sorafenib loaded polymersomes using poly butadiene block poly ethylene oxide (PB-b-PEO), an amphiphilic co-block polymer. Prior to drug loading, critical aggregate concentration (CAC) of polymer was calculated for stable formulation synthesis. The developed SFN loaded PB-b-PEO polymersomes (SFN-PB-b-PEO, test formulation) characterized by DLS and cryo-TEM showed particle size 282 nm, polydispersity (PDI) of less than 0.29 and membrane thickness of about 20 nm. SFN-PB-b-PEO polymersomes demonstrated encapsulation efficiency of 71% and showed sustained drug release up to 144 h. Formulation remained stable for 3 months in suspension form. In vitro cytotoxicity against HepG2 cells showed 1.7 folds improved toxicity compared to SFN suspension. In addition, oral administration of SFN-PB-b-PEO polymersomes in BALB/c mice showed increased Cmax and AUC0-96 by 1.7 and 2.77-fold respectively (p < 0.05) compared to those of SFN suspension (reference formulation). Findings suggest that the SFN-PB-b-PEO polymersomes can be a potential candidate for oral delivery of SFN.

U0126 protects hippocampal CA1 neurons against forebrain ischemia-induced apoptosis via the ERK1/2 signaling pathway and NMDA receptors.

OBJECTIVE: Cerebral ischemia can trigger the ERK1/2 signaling cascade that enables the brain to adapt to ischemic injury. However, the mechanism of ERK1/2 in ischemic brain injury remains unclear. The aim of this study was to examine the roles of the ERK1/2 signaling pathway and NMDA receptors in the apoptosis of CA1 pyramidal neurons after ischemia/reperfusion (I/R). METHODS: Male Wistar rats were subjected to a sham or transient forebrain ischemia procedure. Animals received the intracerebroventricular injection of U0126 (5 µl, 0.2 µg/µl) or vehicle 30 min before ischemia. Homogenates of the hippocampal CA1 field were obtained from sham-operated and ischemic rats 6, 12 or 48 h after ischemia/reperfusion (n = 6 per group) and then subjected to Western blotting analysis and TUNEL staining. Caspase-3 activity was assayed with a colorimetric assay kit. RESULTS: We found that the phosphorylation level of ERK1/2 is increased in the CA1 region following transient I/R. Blocking the ERK1/2 signaling pathway by administration U0126 attenuated apoptotic neuronal cell death via inhibition of NMDA receptors. CONCLUSION: These findings suggest a novel mechanism by which the ERK1/2 signaling pathway affects the post-I/R apoptosis of CA1 pyramidal neurons, which will provide a therapeutic target for the treatment of stroke.

Melatonin exerts an inhibitory effect on insulin gene transcription via MTNR1B and the downstream Raf­1/ERK signaling pathway.

The pineal hormone melatonin influences the secretion of insulin by pancreatic islets via the G­protein­coupled melatonin receptors 1 and 2 that are expressed in pancreatic ß­cells. Genome­wide association studies indicate that melatonin receptor 1B (MTNR1B) single nucleotide polymorphisms are tightly associated with type 2 diabetes mellitus (T2DM). However, the underlying mechanism is unclear. Raf­1 serves a critical role in the mitogen­activated protein kinase (MAPK) pathways in ß­cell survival and proliferation and, therefore, may be involved in the mechanism by which melatonin impacts on T2DM through MTNR1B. In the present study, the mRNA expression of the two mouse insulin genes Ins1 and Ins2 was investigated in MIN6 cells treated with different concentrations of melatonin, and insulin secretion was detected under the same conditions. Following the overexpression or silencing of MTNR1B, the activities of components of the MAPK signaling pathway, including Raf­1 and ERK, were evaluated. The impact of MTNR1B knockdown on the melatonin­regulated insulin gene expression and insulin secretion were also investigated. The results demonstrated that exogenous melatonin inhibited the expression of insulin mRNA in the MIN6 cells. Insulin secretion by the MIN6 cells, however, was not affected by melatonin. The MAPK signaling pathway was inhibited in MIN6 cells by treatment with melatonin or the overexpression of MTNR1B. The knockdown of MTNR1B totally attenuated the regulating effect of melatonin on insulin gene expression. Additionally, the inductive effect of melatonin on the expression of insulin mRNA was attenuated when the activities of Raf­1 or ERK were blocked using the chemical inhibitors GW5074 and U0126, respectively. It may be concluded that melatonin exerts an inhibitory effect on insulin transcription via MTNR1B and the downstream MAPK signaling pathway.

Upregulation of connexin43 by glucose deprivation in H9c2 cells via the extracellular signal­regulated kinase/mitogen­activated protein kinase signaling pathway.

Cardiac connexin43 (Cx43) serves an essential role in maintaining the functional integrity of the heart. The present study investigated the effect of glucose deprivation (GD) on Cx43 protein expression levels in H9c2 cells, and demonstrated that following 2 h GD, Cx43 protein expression levels in H9c2 cells increased by ~68%. In addition, GD activated the extracellular signal­regulated kinase (ERK)/mitogen­activated protein kinase (MAPK) signaling pathway, which regulated the expression levels of cardiac Cx43. A MAPK inhibitor and U0126, an ERK inhibitor, abolished the effects of GD on Cx43 expression levels. Under GD, the protein expression levels of Beclin­1, p62 and LC3 were augmented, and were decreased in the presence of ERK inhibitor or siRNA­ERK. In addition, H9c2 cells exposed to GD exhibited marked increase in LDH release and decreased MTT reduction activity, all of which were not significantly reversed by U0126 treatment. Therefore, the ERK/MAPK signaling pathway may be involved in elevating cardiac Cx43 expression levels under GD in H9c2 cells.

Wild-type p53 controls the level of fibronectin expression in breast cancer cells.

Aberrant fibronectin (FN) expression is associated with poor prognosis, cell adhesion, and cell motility in a variety of cancer cells. In this study, we investigated the relationship between p53 and FN expression in breast cancer cells. Basal FN expression was significantly decreased by treatment with the p53 activator III, RITA, in MCF7 breast cancer cells with wild-type p53. In addition, overexpression of wild-type p53 markedly decreased the level of FN expression in p53-mutant breast cancer cells. To examine the mechanism underlying the relationship between p53 and FN expression, we treated MCF7 breast cancer cells with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). Our results showed that basal FN expression was increased by TPA treatment in a time-dependent manner. In contrast, the level of p53 expression was decreased by TPA treatment. However, the expression of FN and p53 was not altered by TPA in p53-mutant breast cancer cells. Furthermore, the alterations in FN and p53 expression in response to TPA were prevented by a specific MEK inhibitor, UO126. Finally, we demonstrated that TPA triggers degradation of p53 through the proteasomal pathway in MCF7 cells. TPA-induced FN expression was decreased by the proteasome inhibitor MG132. Under the same condition, p53 protein expression, but not mRNA expression, was reversed by MG132. Taken together, our data demonstrate that the level of FN expression is associated with the status and expression of p53 in breast cancer cells.

37LRP induces invasion in hypoxic lung adenocarcinoma cancer cells A549 through the JNK/ERK/c-Jun signaling cascade.

We previously reported that 37-kDa laminin receptor precursor involved in metastasis of lung adenocarcinoma cancer cells. In this study, we further revealed that hypoxia induced 37-kDa laminin receptor precursor expression and activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in lung adenocarcinoma cancer cells. In addition, we further demonstrated that the c-Jun N-terminal kinase inhibitor SP600125 and extracellular signal-regulated protein kinase inhibitor U0126 blocked the c-Jun activity and abolished hypoxia-induced 37-kDa laminin receptor precursor expression and promoter activity in a concentration-dependent manner. However, the p38 mitogen-activated protein kinase inhibitor did not affect 37-kDa laminin receptor precursor expression and c-Jun activity in response to hypoxia. Furthermore, downregulated c-Jun expression by short interfering RNA could also inhibit hypoxia-induced 37-kDa laminin receptor precursor expression and transcriptional activity. The inhibition of 37-kDa laminin receptor precursor expression by SP600125 and U0126 could be rescued by c-Jun overexpression. Studies using luciferase promoter constructs revealed a significant increase in the activity of promoter binding in the cells exposed to hypoxia, which was lost in the cells with mutation of the activator protein 1 binding site. Electrophoresis mobility shift assay and chromatin immunoprecipitation demonstrated a functional activator protein 1 binding site within 37-kDa laminin receptor precursor gene regulatory sequence located at -271 relative to the transcriptional initiation point. Hypoxia-induced invasion of A549 cells was inhibited by the pharmacologic inhibitors of c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated protein kinase (U0126) as well as 37-kDa laminin receptor precursor-specific siRNA or antibody. Our results suggest that hypoxia-elicited c-Jun/activator protein 1 regulates 37-kDa laminin receptor precursor expression, which modulates migration and invasion of lung adenocarcinoma cells.

Transcription factors regulate GPR91-mediated expression of VEGF in hypoxia-induced retinopathy.

Hypoxia is the most important factor in the pathogenesis of diabetic retinopathy (DR). Our previous studies demonstrated that G protein-coupled receptor 91(GPR91) participated in the regulation of vascular endothelial growth factor (VEGF) secretion in DR. The present study induced OIR model in newborn rats using exposure to alternating 24-hour episodes of 50% and 12% oxygen for 14 days. Treatment with GPR91 shRNA attenuated the retinal avascular area, abnormal neovascularization and pericyte loss. Western blot and qRT-PCR demonstrated that CoCl2 exposure promoted VEGF expression and secretion, activated the ERK1/2 signaling pathways and upregulated C/EBP and AP-1. Knockdown of GPR91 inhibited ERK1/2 activity. GPR91 siRNA transduction and the ERK1/2 inhibitor U0126 inhibited the increases in C/EBP ß, C/EBP δ, c-Fos and HIF-1α. Luciferase reporter assays and a chromatin immunoprecipitation (ChIP) assay demonstrated that C/EBP ß and c-Fos bound the functional transcriptional factor binding site in the region of the VEGF promoter, but not C/EBP δ. Knockdown of C/EBP ß and c-Fos using RNAi reduced VEGF expression. Our data suggest that activation of the GPR91-ERK1/2-C/EBP ß (c-Fos, HIF-1α) signaling pathway plays a tonic role in regulating VEGF transcription in rat retinal ganglion cells.

Effects of ERK1/2 signal pathway on cardiomyocyte during glucose lowering.

OBJECTIVE: This study investigated whether the extracellular signal-regulated kinase 1/2 (ERK1/2) signal pathway affects cardiomyocyte apoptosis and the expression of tumor necrosis factor (TNF-α) at different glucose-lowering rates. METHODS: Cardiomyocytes of Wistar neonate rats were maintained in a medium supplemented with 25 mmol/L glucosamine for 72 h. Then the medium was changed to different concentrations of glucosamine, and all cells were divided into five groups. The survival rate of cardiomyocyte was measured using the Cell Counting Kit-8; cardiomyocyte apoptosis was measured using the flow cytometry instrument and laser confocal microscope; TNF-α was measured using the enzyme-linked immunosorbent assay; and ERK1/2 protein and phosphorylation were measured using the Western blot. Cardiomyocyte apoptosis and TNF-α were measured again after adding U0126. RESULTS: As the glucose-lowering rate increased, the survival rate of cardiomyocytes increased in group B and decreased in groups C, D, and E. The TNF-α concentration increased in groups B, C, and D and decreased in group E. After 24 h, the apoptosis rate decreased in group B and increased in groups C, D, and E. The expression of p-ERK1/2 increased in groups B, D, and E, and was the lowest in group C. After adding U0126, the survival rate of cardiomyocyte in all groups increased and TNF-α concentration decreased. CONCLUSIONS: The influence of glucose-lowering rate on cardiomyocyte apoptosis and TNF-α was caused by the p-ERK1/2 pathway. During the glucose-lowering course, the p-ERK1/2 pathway promoted cardiomyocyte apoptosis, and TNF-α secretion was related to not only osmotic pressure but also ERK1/2 signal pathway activation.

Baicalin positively regulates osteoclast function by activating MAPK/Mitf signalling.

Activation of osteoblasts in bone formation and osteoclasts in bone resorption is important during the bone fracture healing process. There has been a long interest in identifying and developing a natural therapy for bone fracture healing. In this study, we investigated the regulation of osteoclast differentiation by baicalin, which is a natural molecule extracted from Eucommiaulmoides (small tree native to China). It was determined that baicalin enhanced osteoclast maturation and bone resorption activity in a dose-dependent manner. Moreover, this involves the activation of MAPK, increased Mitf nuclear translocation and up-regulation of downstream osteoclast-related target genes expression. The baicalin-induced effect on osteoclast differentiation can be mimicked by specific inhibitors of p-ERK (U0126) and the Mitf-specific siRNA, respectively. Protein-ligand docking prediction identified that baicalin might bind to RANK, which is the upstream receptor of p-ERK/Mitf signalling in osteoclasts. This indicated that RANK might be the binding target of baicalin. In sum, our findings revealed baicalin increased osteoclast maturation and function via p-ERK/Mitf signalling. In addition, the results suggest that baicalin can potentially be used as a natural product for the treatment of bone fracture.